WO2025233829A1 - Capsid - Google Patents

Abstract

The present invention relates to an amino acid sequence inserts insertion into an AAV capsid peptide to create a targeted AAV capsid peptide; wherein, when the targeted AAV capsid peptides assemble into a targeted AAV capsid, the targeted AAV capsid has increased tropism for motor neurons relative to an AAV capsid without the insert The invention also relates to full length capsid peptides comprising the inserts, to viral vectors comprising the capsid peptides, and to use of the capsid peptides in viral vectors.

Description

CAPSID

Field of the Invention

The present invention relates to novel AAV capsids with tropism for motor neurons. The invention also relates to amino acid inserts for inclusion into AAV capsid peptides, the inserts increase the tropism of the capsid for motor neurons. The invention further includes viral vectors comprising the capsids, use of the vectors as a delivery vector for gene therapy payloads and for use as a therapeutic. to the Invention

Adeno-associated viruses (AAV) are important vectors for gene therapies. Numerous naturally occurring AAV serotypes have been identified, each with differences in the coding sequence for the viral capsid. These differences in the capsid result in altered cellular tropism, that is, different AAV serotypes will differentially infect different cell types (Castle et al., 2016).

Naturally occurring AAV serotypes have been used in both clinical trials and in approved gene therapies. However, the prevalence of antibodies against natural AAVs in the human population, as well as the inability of natural serotypes to efficiently target certain cell types, means that synthetic (non-wildtype) capsids have become increasingly popular for genetic medicines (Kuzmin et al., 2021).

Synthetic AAV capsids have amino acids sequences that are broadly similar to naturally occurring capsids but are built in the lab via random mutagenesis, DNA shuffling or other synthetic biology techniques. Synthetic capsids can be rationally designed, for example by the insertion of peptide sequences that have potentially useful properties. Alternatively, a large, diverse library of random capsids can be generated (Korbelin et al., 2017). Typical libraries can contain up to 10¹² different variants and a directed evolution approach is used to identify those variants that have desirable properties from this large pool. Desirable qualities include infection of a particular cell type of interest (Bartel et al., 2012), such as neurons, as well as detargeting from other cell types or organs that it is not desirable to target, such as the liver.

Directed evolution of AAV capsids from a library can be done in vivo or in vitro for example, by injecting the library into an animal then harvesting the target tissue or cell type (Figure 1A). If required, this first round of evolution can be supplemented with further rounds, whereby the harvested capsid DNA is further mutated or modified, and the screening process repeated (Grim and Bueng, 2017).

As is generally the case with directed evolution approaches "you get what you screen for". That is to say, the closer that your screening environment replicates the final targeted system, the more likely it is that you will find candidates that possess the desired properties (Schmidt-Dannert and Arnold. 1999). For human gene therapy approaches, it is therefore critical that the screening of AAV capsid libraries occurs in a system that resembles the final human patient as closely as possible. WO2022/013396 and W02024/062450 describe novel methods of directed evolution and screening of AAV capsids that select for tropism for specific cell types. The AAV genome is linear single-stranded DNA (ssDNA) of approximately 4.8 kilobases carrying two ORFs, rep and cap, between inverted terminal repeats (ITRs). Cap encodes overlapping sequences for three capsid proteins VP1, VP2 and VP3 which form the icosahedral capsid of 60 monomers.

All three VPs are translated from one mRNA. After this mRNA is synthesised, it can be spliced in two different manners: either a longer or shorter intron can be excised resulting in the formation of two pools of mRNAs: a 2.3 kb- and a 2.6 kb-long mRNA pool. Usually, especially in the presence of adenovirus, the longer intron is preferred, so the 2.3-kb-long mRNA represents the so-called "major splice". In this form the first AUG codon, from which the synthesis of VP1 protein starts, is cut out, resulting in a reduced overall level of VP1 protein synthesis. The first AUG codon that remains in the major splice is the initiation codon for VP3 protein. However, upstream of that codon in the same open reading frame lies an ACG sequence (encoding threonine) which is surrounded by an optimal Kozak context. This contributes to a low level of synthesis of VP2 protein, which is VP3 protein with additional N terminal residues, as is VP1. Since the bigger intron is preferred to be spliced out, and since in the major splice the ACG codon is a much weaker translation initiation signal, the VP3 peptide if much more prevalent mature viral particles.

Manipulating the cap gene to provide capsid libraries which can be screened for their ability to selectively infect or transduce motor neurons is desirable to develop new gene therapy vectors for treating diseases and conditions associated with dysfunction of motor neurons.

Against this backdrop, the currently disclosed novel capsid peptides with specific tropism for motor neurons have been identified.

Summary of the Invention

According to a first aspect there is provided an amino acid sequence insert according to any one of SEQ ID.l to SEQ ID.24 for insertion into an AAV capsid peptide to create a targeted AAV capsid peptide; wherein, when the targeted AAV capsid peptides assemble into a targeted AAV capsid, the targeted AAV capsid has increased tropism for motor neurons.

Increased or improved tropism is relative to the same AAV capsid without the insert. Tropism can be measured, for example, by comparing the number or proportion of viral particles that transduce motor neurons.

In one embodiment the tropism for motor neurons is tropism for motor neuron synaptic terminals.

In a second aspect there is provided the use of an amino acid sequence insert according to any one of SEQ ID.l to SEQ ID.24 as an insertion into an AAV capsid peptide to generate a targeted AAV capsid peptide which assembles into a targeted AAV capsid with increased tropism for motor neurons.

In a third aspect there is provided a targeted AAV capsid peptide comprising the amino acid sequence insert according to any one of SEQ ID.l to SEQ ID.24 inserted into an AAV capsid peptide.

In one embodiment the AAV capsid peptide is an AAV1, AAV2, or AAV6 serotype capsid peptide.

In one embodiment the AAV capsid peptide has the amino acid sequence according to any one of SEQ ID.50 to SEQ ID.52. In one embodiment the targeted AAV capsid peptide according to the present disclosure comprises an amino acid sequence insert inserted at position, Q585/S586 or S588/T589 in SEQ ID.50, N587/R588 in SEQ ID.51, or Q585/S586, S586/S587, or S587/S588 in SEQ ID.52 or the equivalent position thereof.

In one embodiment the insert is at positions 585/586 of AAV1.

In one embodiment the insert is at position 588/589 of AAV1.

In one embodiment the insert is at position of 587/588 of AAV2.

In one embodiment the insert is at position 585/586 of AAV6.

In one embodiment the insert is at position 586/587 of AAV6.

In one embodiment the insert is at position 587/588 of AAV6.

In one embodiment the AAV capsid peptide is wild type AAV capsid peptide.

In one embodiment the AAV capsid peptide is VP1.

In one embodiment the AAV capsid peptide is VP2.

In one embodiment the AAV capsid peptide is VP3.

In one embodiment the targeted AAV capsid peptide has the amino acid sequence according to any one of SE ID.53 to SEQ ID.90.

In a fourth aspect there is provided a targeted AAV capsid comprising one or more targeted AAV capsid peptides according to the present disclosure.

In a further aspect there is provided targeted AAV viral vector comprising the targeted AAV capsid according to the present disclosure and a nucleotide-encoded payload.

In one embodiment the targeted AAV capsid carries a nucleotide-encoded payload to provide a targeted AAV viral vector.

In one embodiment the nucleotide-encoded payload encodes a therapeutic peptide.

In one embodiment the nucleotide-encoded payload is a gene therapy payload.

In a further aspect there is provided the use of the AAV viral particles disclosed herein in the treatment of a disease caused by a genetic mutation.

Advantageously, the AAV capsids described herein may be used to develop gene therapies for treating various conditions or disorders. Accordingly, the AAV capsids may be employed in a method of ameliorating or treating a neuromuscular or neuromotor condition or disorder in a subject, comprising administering to the subject a therapeutically active amount of an AAV expression vector or viral particle of the invention.

A yet further aspect provides a nucleotide sequence encoding the amino acid sequence according to any one of SEQ ID.l to SEQ ID.24 or SEQ ID.53 to SEQ ID.90. The nucleotide sequence may be used in the generation of a targeted AAV capsid with increased tropism for motor neurons. A further aspect provides an AAV capsid peptide fragment for improving the specificity of transduction of motor neurons, the fragment comprising the amino acid sequence of SEQ ID.1 to SEQ ID.24.

Another aspect provides an AAV capsid capable of transducing a human motor neuron comprising the amino acid sequence according to any one of SEQ ID.l to SEQ ID.24 or SEQ ID.53 to SEQ ID.90.

Also provided is a recombinant AAV (rAAV) vector comprising a capsid, the capsid comprising peptides containing the amino acid sequence according to any one of SEQ ID.l to SEQ ID.24 or SEQ ID.53 to SEQ ID.90.

Brief Description of the Drawings

For a better understanding of the invention and to show how the same may be carried into effect, there will now be described by way of example only, specific embodiments, methods and processes according to the present invention with reference to the accompanying drawings in which:

Figure 1 shows an example of directed evolution and screening as employed in prior art methods. At 1 and 2, cells (e.g. iPSCs) are grown into neurons. At 3, capsids from the AAV library containing marker DNA (e.g. the nucleotide sequence encoding the capsid) are introduced to the terminals ends of the neurons, the cell bodies are distally removed and isolated from the "infection/transduction" site. At 4 the cell bodies are harvested and marker DNA from the capsids that successfully transduced the neurons is sequenced. At 5 new capsids are generated from the successful capsids in the previous round and a further round of directed evolution occurs. Typically, 3-5 rounds of directed evolution are carried out.

Figure 2 shows in vitro testing of lead capsid candidates and comparison to parental wildtype serotypes using a luciferase assay in human derived motor neurons. (A) AAV2-based candidate comparison, (B) AAVl-based candidates compared to AAV1 control, (C) AAV6 based candidates compared to control.

Figure 3 shows examples of fluorescence expression with AAV2 WT (SEQ ID.51) or SEQ ID.53, SEQ ID.56 and SEQ ID.55.

Figure 4 shows in vivo testing of capsids in mouse spinal cord compared to WT AAV2 capsid (SEQ ID.51). The capsids contained a marker payload in the form of mScarlet and were injected into the gastrocnemius muscle. The spinal cords were stained with ChAT to show where motor neurons were located, transduced motor neurons expressed mScarlet. AAV2 does not localise to the motor neurons, SEQ ID.65 and SEQ ID.54 are clearly seen to localise in the motor neurons.

The bar charts show the presence of capsid in the lumbar spinal cord segment. SEQ ID. is found in the spinal cord, indicating retrograde transport from the axon terminals located in the muscle to the spinal cord. AAV2 (as a control) was not found. In this example, red fluorescent protein (RFP) was only present when SEQ ID.65 was used, not when WT AAV2 was used.

Figure 5 shows detargeting of the presently disclosed capsid in different cell types. Capsids carrying mScarlet were only found in the gastrocnemius (leg muscle) showing that off target effects are unlikely. Within the gastrocnemius the proportion of various SEQ ID capsids was tested and found to be primarily made up of WT AAV, the other capsids having transduced the motor neurons and therefore left the muscle, or been broken down over time if they failed to transduce any cells.

Figure 6 shows protein modelling of the WT capsid peptide (6A) with the insertion site indicated by *. 6B shows the insertion of SEQ ID.l as present in SEQ ID.53 and 6C shows the insertion of SEQ ID.l in an alternative location (residues 454/455).

Figure 7 shows in vivo testing of capsid SEQ ID.53 in mouse spinal cord. The capsid contained a marker payload in the form of GFP and were injected into the gastrocnemius muscle. The spinal cords were stained using RNAScope technique with ChAT to show where motor neurons were located, transduced motor neurons expressed GFP. SEQ ID.53 is clearly seen to localise in the motor neurons. GFP signal can be observed in dorso-medial range which corresponds to range expected for gastrocnemius motor column, proving the specificity of SEQ ID.53.

Figure 8 shows detargeting of capsids in different cell types compared to AAV6 WT (SEQ ID.52). GFP signal was detected at DNA level at minimal levels in the injected muscle of capsids SEQ ID.53, 54 and 55 compared to WT control (Figure 8A). Minimal to none GFP VCN were found in non-injected muscle and liver. GFP mRNA expression quantification followed a similar pattern. Although levels were minimal in non-injected muscle, GFP mRNA expression was higher in AAV6 WT (SEQ ID.52) capsid in the injected muscle compared to capsids SEQ ID.53, 54 and 55, proving detargeting from the injected muscle.

Figure 9 shows biodistribution of capsids SEQ ID.53 and SEQ ID.54 when injected intravenously. Figure 9A shows a very clean profile if these capsids were to be administered iv, with minimal VCN found in the liver, as expected for this route of administration. Figure 9B shows number of transduced (GFP + cells ) motor neuron (ChAT+ cells) quantified in cervical and lumbar segments of the spinal cord for each given capsid.

Detailed Description

The presently disclosed capsids were generated using proprietary directed evolution and screening methods to develop and identify novel capsids with beneficial tropism for motor neurons and other properties. Screening refers to the method of selectively identifying members of a population for desirable properties. Herein, the population is an AAV library, or AAV viral particle library, and the desirable property is the ability to selectively infect (transduce) motor neurons.

As employed herein amino acid sequence insert (or, insert) refers to a sequence of amino acids typically 7 to 27 residues long and identified by SEQ ID. 1 to 24. The amino acid sequences are known to have beneficial properties when inserted into AAV capsid peptides. For example, into AAV1, AAV2, or AAV6 capsid peptides as identified in SEQ ID. 50 to SEQ ID.52. The inserts are known to improve, or increase, tropism for motor neurons. The insert sequences may be tolerant of sequence changes (substitutions, deletions, insertions) without affecting the tropism for motor neurons.

In one embodiment the amino acid sequence insert has the sequence of any one of SEQ ID. 1 to SEQ ID.24, or an equivalent sequence maintaining the properties thereof. In one embodiment the amino acid sequence insert has the sequence of any one of: SEQ I D.1, SEQ ID.5, SEQ I D.6, SEQ. I D.7, SEQ ID.8, SEQ I D.9, SEQ ID.10, SEQ ID.11, SEQ I D.12, SEQ I D.13, SEQ ID.14, SEQ ID.15, SEQ I D.16, SEQ ID.17, SEQ I D.18, SEQ I D.19, SEQ ID.20, SEQ ID.21, SEQ I D.22, SEQ ID.23, and SEQ ID.24.

In one embodiment the amino acid sequence insert has the sequence of SEQ I D.l.

In one embodiment the amino acid sequence insert has the sequence of SEQ ID.2.

In one embodiment the amino acid sequence insert has >90% sequence identity to SEQ ID.l to SEQ ID.24, such as 91, 92, 93, 94, 95 96, 97, 98 or 99% sequence identity to SEQ ID.l to SEQ ID.24.

As employed herein AAV capsid peptide is an untargeted AAV capsid peptide and typically means a wild type AAV capsid peptide.That is, an individual VP1, VP2 or VP3 peptide that is unmodified. Full length WT AAV capsid peptide sequences are indicated, for example, by SEQ. ID.50 to SEQ ID.52.

In one embodiment the AAV capsid peptide has the sequence of any one of SEQ ID.50 to SEQ ID.52. It will be appreciated that fragments of the AAV capsid peptide and AAV capsid peptides with different sequences may still form capsids falling within the scope of the present invention if they have the inserts disclosed herein. The AAV capsid peptide sequence may have >80% sequence identity to SEQ ID.50 to SEQ ID.52, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 96, 97, 98 or 99% sequence identity to SEQ ID.50 to SEQ ID.52.

As employed herein targeted AAV capsid peptide refers to AAV capsid peptides comprising the insert of SEQ ID.l to SEQ ID.24, such that the targeted AAV capsid peptide forms a capsid with the ability to selectively transduce motor neurons. The specificity may be motor neurons in preference to muscle cells, for example. Alternatively, it may be motor neurons in preference to sensory neurons, for example. In general, detargeting away from non-motor neuron cell types is desirable.

In one embodiment the targeted AAV capsid peptide comprises the amino acid sequence insert according to any of SEQ ID.l to SEQ ID.24.

In one embodiment the targeted AAV capsid peptide comprised the amino acid sequence insert according to SEQ ID. 1.

In one embodiment the targeted AAV capsid peptide comprised the amino acid sequence insert according to SEQ ID. 2.

In one embodiment the targeted AAV capsid peptide comprises or consists of the amino acid sequence according to any of SEQ ID.53 to SEQ I D.90. In one embodiment the targeted AAV capsid peptide comprises the amino acid sequence according to any of SEQ ID.53, SEQ ID.54, SEQ ID.55, SEQ ID.56, SEQ ID.57, SEQ ID.58, SEQ ID.59, SEQ ID.60, SEQ ID.62, SEQ ID.63, SEQ ID.64, SEQ ID.66, SEQ ID.67, SEQ ID.68, SEQ ID.69, SEQ ID.70, SEQ ID.71, SEQ ID.72, SEQ ID.73, SEQ ID.74, SEQ ID.75, SEQ ID.76, SEQ ID.77, SEQ ID.78, SEQ ID.79, SEQ ID.83, SEQ ID.84, SEQ ID.85, SEQ ID.87, or SEQ ID.89.

In one embodiment the targeted AAV capsid peptide comprises the amino acid sequence according to any of SEQ ID.53, SEQ ID.54, SEQ ID.55, SEQ ID.56, SEQ ID.57, SEQ ID.58, SEQ ID.62, SEQ ID.64, SEQ ID.65, SEQ ID.70, SEQ ID.71, SEQ ID.76, SEQ ID.78, SEQ ID.79, SEQ ID.80, or SEQ ID.84. In one embodiment the targeted AAV capsid peptide comprises or consists of the amino acid sequence according to SEQ ID.53.

In one embodiment the targeted AAV capsid peptide comprises or consists of the amino acid sequence according to SEQ ID.54.

In one embodiment the targeted AAV capsid peptide comprises or consists of the amino acid sequence according to SEQ ID.55.

AAV capsid as employed herein refers to the capsid of adeno-associated viruses. Herein, AAV capsids are comprised of AAV capsid peptides assembled into a functional capsid. AAV capsids are unmodified, non-targeted AAV capsids. That is, the capsid peptides do not comprise the inserts or mutations disclosed herein. AAV capsids become targeted AAV capsids when they comprise capsid peptides comprising the inserts or mutations disclosed herein (i.e. targeted capsid peptides).

It is known that the AAV capsid is capable of remarkable selectivity due to its make up (the AAV capsid is composed of a mixture of VP1, VP2, and VP3 totalling 60 monomers arranged in icosahedral symmetry in a ratio of 1:1:10) and post-translational modifications. AAV capsid proteins contain 12 hypervariable surface regions, but the genome, in general, presents highly conserved replication and structural genes across serotypes.

Serotype as employed herein refers to standard nomenclature, AAV1, AAV2, AAV6 etc, of the wild-type capsid prior to mutation (by insertion or point mutation) as disclosed herein. In the wild, multiple serotypes of AAV have been identified each with unique sequences of capsid gene, and hence distinct tropisms, although wild serotypes tend to be able to infect multiple tissue and cell types. These serotypes are denoted by numbers: AAV1, AAV2, etc. It has been shown that modification of capsid sequences via DNA recombination methods can generate non-native sequences with tailored properties and tropism directed towards (or against) particular cells or tissues, and that evade the immune system (Vandenberghe et al., 2009).

In one embodiment the AAV capsid is an AAV1, AAV2, or AAV6 serotype capsid.

In one embodiment the AAV capsid is comprised of AAV capsid peptides according to SEQ ID.50 to SEQ ID.52.

As employed herein targeted AAV capsid refers to an AAV capsid comprising one or more targeted AAV capsid peptides. Targeted AAV capsids selectively infect or transduce motor neurons, such as a targeted AAV capsid disclosed herein. Typically, targeted AAV capsids comprise peptides that are predominantly targeted AAV capsid peptides. In general, WT AAV capsid peptides are not present in targeted AAV capsids, however, a proportion of WT peptides is not precluded from consideration provided their presence does not reverse or hinder the increased tropism for motor neurons.

As employed herein increased tropism refers to the property shared by the disclosed targeted capsids whereby the capsid can infect/transduce motor neurons more than their equivalent WT capsid. Viral tropism is the ability of different viruses to infect different cellular types ultimately to produce a successful infection. The effectiveness of an AAV particle (capsid) to transduce/infect specific neurons can be determined by counting the number of neurons that express the viral DNA, such as GFP. For example, multiple motor neurons innervate the same targets, e.g. muscle in the case of motor neurons, and the proportion of these neurons that have been transduced by the AAV viral particle can be counted. The effectiveness of an AAV viral particle to infect neurons or specific parts of neurons can also be determined by DNA sequencing or RT- PCR to look at the "copy number" of the viral DNA that is in the neural cell. This would give an estimate of how many times the same cell was infected with the AAV viral particle. Other methodologies may be apparent to the skilled person.

Capsids disclosed herein (targeted AAV capsids) have specific tropism for motor neurons and selectively transduce motor neurons. Capsids disclosed herein display increased motor neuron transduction relative to WT AAVs of the same serotype (including variants that are not necessarily the canonical WT sequence but do not comprise the inserts or mutations disclosed herein) and relative to other capsids screened during directed evolution. Capsids disclosed herein display increased transduction of motor neurons in vitro and in vivo. Capsids disclosed herein display limited transduction of cell types that are not motor neurons. Capsids disclosed herein are detargeted away from non-motor neuron cell types.

In some embodiments the targeted AAV capsid may also be detargeted from infecting or transducing other (non-motor neurons) cell types. Such properties are beneficial to avoid undesirable off-target effects during gene therapy or chemogenetics therapy.

Detargeted refers to the reduction or removal of the virus to be transported to and/or to infect other cell types. In the present disclosure, it is undesirable for the capsids to travel to and infect cells other than motor neurons. For example, if muscle cells were transduced that would reduce the amount of AAV available to transduce the motor neurons and may lead to off-target effects. In general, detargeting is beneficial in reducing the body's immune response to the virus.

The term neuron includes a neuron and a portion or portions thereof (e.g., the neuron cell body, an axon or a dendrite). The term neuron as used herein denotes nervous system cells that include a central cell body (or soma) and two types of extensions or projections: dendrites, by which the majority of neuronal signals are conveyed to the cell body, and axons, by which the majority of neuronal signals are conveyed from the cell body to effector cells, such as target neurons or muscle

As employed herein, neurons refers to motor neurons.

Use of the amino acid sequence inserts disclosed herein to increase tropism for motor neurons means that the inserts of SEQ ID.l to SEQ ID.24 can be inserted into AAV capsid peptides to make the new (targeted) capsid peptide with tropism for motor neurons. In some cases, the tropism for motor neurons may be a new property. In other cases, the tropism for motor neurons may be increasing or improving an existing property of the capsid peptide.

In some embodiments the amino acid sequence inserts (the inserts) are inserted into specific locations in the AAV capsid peptide sequence. As described herein the locations are referred to in reference to the sequences shown in SEQ ID.50 for AAV1, SEQ ID.51 for AAV2 and SEQ ID.52 for AAV6. Certain of the capsids disclosed herein, where the serotype is AAV1, contain insertions at Q585/S586 or S588/T589. Certain of the capsids disclosed herein, where the serotype is AAV2, contain insertions at N588/R589. Certain of the capsids disclosed herein, where the serotype is AAV6, contain insertions at Q585/S586, S586/S587, or S587/S588. The nomenclature used refers to the two residues between which the insert is inserted.

In one embodiment the serotype is AAV1, and the amino acid sequence insert is inserted at Q585/S586 or S588/T589 of SEQ ID.50, or the equivalent position in a variant thereof.

In one embodiment the serotype is AAV2, and the amino acid sequence insert is inserted at N587/R588 of SEQ ID.51, or the equivalent position in a variant thereof.

In one embodiment the serotype is AAV6, and the amino acid sequence insert is inserted at Q585/S586, S586/S587, or S587/S588 of SEQ ID.52, or the equivalent position in a variant thereof.

In one embodiment insert SEQ ID. 1 is inserted at N587/R588 of an AAV2 serotype capsid peptide. For example, in SEQ ID. 51 or at the equivalent position in a variant thereof.

In one embodiment insert SEQ ID. 1 is inserted at S588/T589 of an AAV1 serotype capsid peptide. For example, in SEQ ID. 50 or at the equivalent position in a variant thereof.

In one embodiment insert SEQ ID. 2 is inserted at N587/R588 of an AAV2 serotype capsid peptide. For example, in SEQ ID. 51 or at the equivalent position in a variant thereof.

As employed herein AAV viral vector refers to adeno-associated virus viral particles comprising a single stranded DNA genome packaged within a capsid as disclosed herein. In particular, the AAV viral vectors employed herein may encode a therapeutic payload. Target AAV viral vectors are those comprising a targeted AAV capsid.

In some embodiments the viral vectors may be devoid of replication-encoding nucleotides, that is, they are replication deficient.

Nucleotide-encoded payload as employed herein refers to exogenous transgene(s) that may be delivered to a motor neuron using the targeted viral vectors disclosed herein. Advantageously, this payload can be used to deliver gene therapy to targeted cells. Typically, the payload or transgene will encode a therapeutic peptide. That is, a peptide that is either deficient or erroneously expressed in the target cell(s).

In one embodiment the targeted AAV viral vectors can be used in gene therapy methods to deliver exogenous transgenes to a subject in need thereof.

The gene therapy may be used to treat a neurological disorder or condition associated with motor neurons. One example of a neurological disorder or condition that can be treated by the gene therapy is spasticity. Spasticity is a neurological symptom suffered by people with a variety of neurological disorders, including but not limited to multiple sclerosis, stroke, traumatic brain injury, spinal cord injury, and cerebral palsy. Spasticity results from excessive excitation of muscle by motor neurons, which, because of the disease, become "hyperexcitable".

In one embodiment, the targeted AAV viral vector further comprises a transgene encoding a transgene product, wherein the transgene product is capable of altering the activity motor neurons in a subject. In one embodiment, the transgene product is capable of altering the activity of motor neurons in a subject via intramuscular injection.

In one embodiment, the payload is nucleotide sequence encoding a Kv7.3 ion channel subunit. The ion channel subunit may be a wild type or a mutant.

In use, the targeted AAV viral vector containing the nucleotide sequence encoding a Kv7.3 ion channel is delivered to a subject, the vector infects motor neurons which become transduced neurons. The transduced neurons overexpress the amino acid sequences encoded by the nucleotide sequences, which assemble as functional Kv7.3-containing ion channels (either homomers or heteromers). In general, the time from delivery of the viral particles to the subject to overexpression of the functional ion channels may be approximately 3 weeks. Once a suitable period of time has elapsed for the transduced neurons to over express the ion channels, the subject could be treated with a neuromodulatory drug, such as Retigabine, at a lower dose.

The invention also comprises methods of treatment which involve injecting targeted AAV viral vectors comprising an exogenous transgene as described herein into affected muscles of a subject; these AAVs can then transduce the motor neurons, and are transported to their cell bodies leading to the expression of an exogenous transgene specifically in the motor neurons innervating that muscle, providing high specificity.

The invention can therefore enable the generation of targeted AAV viral vectors that access motor neurons and subsequently modify gene expression in the motor neurons with the goal of curing, alleviating symptoms, and/or improving the quality of life of patients with diseases affecting, or caused by dysfunctional, motor neurons.

The invention provides targeted AAV viral vectors that access motor neurons following, for example, intramuscular injection, and subsequently modify gene expression in these neurons with the goal of curing, alleviating symptoms, and/or improving the quality of life of patients with diseases affecting motor neurons.

In some embodiments, the targeted AAV viral vector retrogradely transduces motor neurons with the purpose of treating neuromuscular or neuromotor disorders, or disorders affecting movement.

In some embodiments, the targeted AAV viral vector is delivered intramuscularly, in order to infect motor neurons of a subject neuron retrogradely and alter the activity of the motor neurons in a subject.

As used herein, "retrograde transport" or "retrograde infection" means uptake of the vector at the axon terminal (or "synaptic terminal"), i.e., at the synaptic portion, and transport through the axon in a direction opposite to the direction of propagation of action potentials (and thus "retrograde") and into the body of the neuron. Subsequently, the viral nucleic acid can enter the nucleus where it can be replicated and become transcriptionally and translationally active.

Such delivery is advantageous when the neuronal cell body and or axon themselves are inaccessible, but their terminal projection fields including synapses, are available for delivery of the genetic vector. Successful delivery to such a terminal projection field of a genetic vector capable of retrograde transport would thus result in retrograde transport and infection of the vulnerable projection neurons. Once the viral vector is transported to the body of the neuron, the viral nucleic acid typically localises to the nucleus of the cell. According to some embodiments of the present invention, adeno-associated viral vectors that undergo retrograde transport to the neuronal body can insert their nucleic acid content directly into the nucleus.

The targeted AAV capsids disclosed may be used to develop gene therapies involving viral vectors that access neurons following intramuscular injection, and subsequently modify activity and/or gene expression in neurons.

In some embodiments, the targeted AAV viral vector is capable of altering the activity of motor neurons in a subject, for example, via intramuscular injection.

Improving specificity for transduction as employed herein refers to one or more of the following:

Increasing, improving or introducing tropism to motor neurons, and Detargeting non-motor neuron cell types

In one embodiment the amino acid sequence inserts of SEQ ID.l to SEQ ID.24 can be used to improve the specificity of an AAV capsid for transducing motor neurons.

Capable of transducing as employed herein means that the AAV capsid or viral vector can infect a particular cell type (motor neurons) and hence can deliver nucleic acid to the cell.

In one embodiment the targeted AAV capsids disclosed herein are capable of transducing motor neurons.

In one embodiment some of the capsids disclosed herein may further comprise point mutations, or contain only point mutations and no amino acid sequence insert. Examples of such capsids are shown in SEQ ID.76, SEQ ID.77 and SEQ ID.89. In each of the foregoing examples, the mutations of AAV6 K531E, L584F, V598A are mutations to residues seen in AAV1 capsids. Residue numbers are in reference to SEQ ID.52. Without wishing to be bound by theory, it is thought that making the capsid more AAVl-like by introducing the point mutations may influence the tropism of the AAV6 capsid.

In one embodiment the targeted AAV capsid peptide does not comprise an insert but does comprise point mutations according to SEQ ID. 76, SEQ ID.77 or SEQ ID.89.

The invention also extends to nucleotide sequences encoding the amino acid sequence of the insert and/or the capsid peptide. These can be referred to as insert-encoding nucleotide sequences or capsidencoding nucleotide sequences respectively. Insert-encoding and capsid-encoding nucleotide sequences refers to nucleotide sequence encoding the insert peptide or capsid peptide respectively. Typically, the nucleotide sequence is a DNA sequence, although RNA, modified nucleotide and synthetic nucleotides are also envisioned.

In one embodiment the insert-encoding nucleotide comprises the nucleotide sequence of any one of SEQ ID.25 to SEQ ID.49.

In one embodiment the insert-encoding nucleotide comprises the sequence of any one of: SEQ ID. 26, SEQ ID. 27, SEQ ID.28, SEQ ID.31, SEQ ID.32 to SEQ ID.45, SEQ ID.47, SEQ ID.48, and SEQ ID.49. In one embodiment there is provided a rAAV vector comprising a capsid, the capsid comprising peptides containing the amino acid sequence according to any one of SEQ. ID.1 to SED ID.24 or SEQ ID.53 to SEQ ID.90.

Recombinant AAV (rAAV) vectors developed using the capsids described herein are especially applicable to the treatment of neuromuscular/neuromotor disorders such as spasticity, amyotrophic lateral sclerosis, dystonia, allowing for the introduction of genetic material into motor neurons via intramuscular injection of viral vectors.

As employed herein a "neuromotor disorder" is a developmental or acquired disorder that typically affects movement/gross motor ability, posture, and fine motor ability. The disorder is caused by damage to the central nervous system. This could be due to problems with development or injury to the developing motor pathways in the cortex, basal ganglia, thalamus, cerebellum, brainstem, spinal cord, or peripheral nerve. The most common neuromotor disorders in childhood include cerebral palsy, muscular dystrophy, and spina bifida. The most common neuromotor disorders in adults include stroke, multiple sclerosis, Parkinson's disease and traumatic injury. The impairment may be static (not getting worse) or progressive.

The invention also provides a cell comprising a targeted AAV viral vector as described herein. In some embodiments, this cell is a mammalian cell such as a human cell.

A subject as employed herein means a human or non-human mammal. Typically, the subject has a neurological disorder associated with increased excitability of neurons.

In some embodiments the subject has a neurological disorder associated with dysfunction of Kv7.3 ion channels.

In some embodiments the subject has symptoms characterised by neuronal hyperexcitability that may or may not be associated with dysfunction of Kv7.3 ion channels.

In some embodiments the subject has a neurological disorder and/or symptoms characterised by neuronal hyperexcitability that may or may not be associated with dysfunction of Kv channels.

Neurological disorder associated with increased neuronal excitability as employed herein refers to any condition in which excess neuronal activity leads to symptoms associated with the same neurological disorder. For example, epilepsy, spasticity, benign familial neonatal seizures (BNFS), nociceptive pain, non-nociceptive pain, Parkinson's disease, multiple sclerosis.

In one embodiment the neurological disorder is associated with hyperexcitability of motor neurons.

In one embodiment the neurological disorder is associated with increased excitability of motor neurons.

In one embodiment the neurological disorder is associated with motor neuron degeneration or motor neuron death.

In one embodiment the neurological disorder is associated with hypoexcitability of motor neurons. In one embodiment the neurological disorder is associated with decreased excitability of motor neurons.

In one embodiment the neurological disorder is selected from epilepsy, spasticity, BNFS, nociceptive pain, non-nociceptive pain, Parkinson's disease, multiple sclerosis.

In one embodiment the neurological disorder is spasticity.

Treating or treatment as employed herein refers to the reversal of a condition, amelioration or relief of symptoms associated with a condition or prevention of further development/worsening of a condition.

The term "treatment" includes combination treatments and therapies, in which two or more treatments or therapies are combined, for example, sequentially or simultaneously. For example, the vector may be administered a day or more before the neuromodulatory drug is administered to the subject.

In one embodiment the targeted AAV capsid peptide is selected from the group consisting of SEQ ID.53 to SEQ ID.90 or an amino acid sequence with >90% sequence identity thereto.

In one embodiment the nucleotide sequence comprises the sequence of SEQ ID.25 to SEQ ID.49 or a variant thereof encoding the same amino acid sequence.

In one embodiment the targeted AAV capsid peptide comprises or consists of the amino acid sequence according to any one of SEQ ID.53 to SEQ ID.90.

In the context of this specification "comprising" is to be interpreted as "including".

Aspects of the invention comprising certain elements are also intended to extend to alternative embodiments "consisting" or "consisting essentially" of the relevant elements.

Where technically appropriate, embodiments of the invention may be combined.

Embodiments are described herein as comprising certain features/elements. The disclosure also extends to separate embodiments consisting or consisting essentially of said features/elements.

Approximately as employed herein is intended to mean ±10%.

Technical references such as patents and applications are incorporated herein by reference.

Any embodiments specifically and explicitly recited herein may form the basis of a disclaimer either alone or in combination with one or more further embodiments.

Examples

Library generation, screening and directed evolution

Modifications to capsids can be achieved in several ways, such as random mutagenesis of existing capsid DNA sequences, by capsid shuffling (i.e. taking DNA sequences from multiple capsids and randomly shuffling parts of the sequence to make a new capsid; Buning et al., 2015), or by insertion of short peptide sequences into the exposed loop regions of the capsid. These methods can generate a large number of highly diverse capsids with potentially valuable properties. When packaged into functional virions, they can be screened in animal tissues or in cell cultures to select those capsids that are targeted towards particular tissues or cells. These capsid sequences can then be generated de novo and combined with genes with therapeutic potential in a gene therapy.

In vivo studies have shown that directed evolution of AAV capsids can lead to vectors that have useful properties such as an ability to cross the blood brain barrier and increased neurotropism (Deverman et al., 2016 and EP3044318B1), or directed towards dopaminergic neurons (Daviddson et al., 2019) or cardiomyocytes (Yang et al., 2009) amongst others (for review see Li and Sumulski, 2020). Examples of directed evolution include the generation of a capsid library, a mixture of AAV vectors encapsidated with random capsid sequences generated via error prone PCR, capsid shuffling, or other methods. These libraries have been applied to cell lines (e.g. Maheshri et al., 2006), undifferentiated stem cells (Asuri et al., 2012) or, most commonly, in experimental animals (for examples Devermann et al., 2016; Li and Sumulski, 2020; Patents US8632764B2; US20170166926A1; US9701984B2).

Diverse capsid libraries can be generated through a process of, for example: i) random mutagenesis of naturally occurring capsids, ii) shuffling of naturally occurring capsids, iii) insertion of targeted or random peptide sequences up to 25 amino acids in length at various regions in VP1, VP2 or VP3 of the AAV capsid or iv) a combination of the above.

Methods for production of AAV libraries comprise one more of the following steps:

-Purified and concentrated AAV libraries are diluted in Dulbecco's modified Eagle medium (DMEM), and this is applied to the muscle chamber of a microfluidic device.

-2-10 days after application neuronal cell bodies are harvested either by chemical (i.e. trypsinisation) or mechanical (cell scraping) methods.

-The neurons are lysed and the lysate can either be submitted for deep sequencing (such as RNAseq or DNAseq) to directly detect capsid sequences found in the neurons, or the lysate can be used as a PCR template using primers directed against conserved regions of the AAV capsid. Following PCR of capsid regions, the DNA fragment is cloned into a DNA vector and submitted for Sanger sequencing.

-Capsid sequences harvested from neurons are analysed using bioinformatics for conserved regions and highly enriched capsids can be de novo synthesised and can then undergo either further mutagenesis or repeat of in vitro screening to increase evolutionary pressure through directed evolution.

-Directed evolution can be repeated for several rounds (~2-5 rounds). Capsid sequences that show efficient retrograde transport in vitro can be used to generate functional virions for in vivo use.

To generate the library the randomised capsid sequences are cloned into an AAV backbone containing the AAV2 inverted terminal repeats (ITRs; packaging signals) and the AAV2 rep gene. These DNA plasmids are transfected into HEK293 in the presence of additional adenoviral genes to facilitate AAV packaging. AAV virions are harvested from HEK293 cells and/or the culture medium, purified and concentrated following standard methods (e.g. Potter et al., 2014 https://dx.doi.ora/10.1038%2Fmtm.2014.34; McClure et al., 2011 htp://dx.doi.Org/10.3791/3348)

Capsid with payload

Methods for the addition of a payload to alter neuron activity may include one or more of the following steps:

-Capsid sequences identified in the screening methods described herein are de novo synthesised and inserted into an AAV helper plasmid containing the AAV2 REP gene (Rep/Cap).

-The Rep/Cap plasmid is combined with the AAV backbone and additional plasmids containing adenoviral helper genes (such as pHelper) and transiently transfected into HEK293 cells.

-AAV particles are purified using standard methods and can be used for experiments in vitro or in vivo (e.g. Potter et al., 2014 https://dx.doi.org/10.1038%2Fmtm.2014.34: McClure et al., 2011 http://dx.doi.org/10.3791/3348)-

Human MN transduction

Human motor neurons were generated from a pooled hIPSC cell line using published methods (Maury et al 2014) with some modifications.

Each selected capsid candidate was packaged with dual reporters that allow fluorescence detection by mScarlet reporter and luciferase activity quantification.

Candidates were tested in vitro for their ability to transduce human motor neurons with a MOI of 30,000. One week after AAV addition Luciferase Assay was performed which allows to quantify reporter protein expression. Luciferase activity was normalised for each candidate by calculating the foldchange compared to control activity. (The graphs show foldchange; N=3-4, Average +- SEM).

Human Neuromuscular organoids transduction

Human trunk neuromuscular organoids were generated from a pooled hIPSC cell line using published methods (Martins et al 2020). Candidates were tested in vitro for their ability to transduce human neuromuscular organoids with a MOI of 30,000. Two weeks after AAV addition we performed Luciferase Assay which allows to quantify reporter protein expression. We normalised the luciferase activity of each candidate by calculating the foldchange compared to control activity. (The graphs show foldchange; N=2, Average +- SEM).

Methods for in vivo validation of the capsid

Each selected capsid candidate was packaged with dual reporters that allow fluorescence detection by mScarlet reporter and luciferase activity quantification.

Those capsids that had a good performance in vitro were moved to in vivo validation. We injected the selected candidates in adult black mice (8-10 weeks). The AAVs were injected bilaterally with a volume of 10 pl at each side under general anaesthesia. For each injection, we performed three x 3.3ul deposits per injection in 4mm depth at 1.3mm intervals. Because of the angle and the depth required, the whole triceps surae (calf) will be targeted, which includes the gastrocnemius lateral and medial, and the soleus muscles.

For motor neuron transduction efficiency, four weeks after AAV injection mice were perfused, and spinal cord was collected. We performed immunofluorescence for general motor neuron marker (Choline acetyltransferase, ChAT) and mScarlet (our AAV reporter). We carried out quantification of the positive cells for each marker in the L3-L6 lumbar spinal cord segments.

Methods around the selection of the capsid candidates to be validated in vitro and in vivo

To analyse evolution data, following DNA extraction we perform PCR with a low number of amplification cycles.

This amplifies the region of the capsid that contains the variant with minimal bias introduced by PCR. These samples are submitted to short read next generation sequencing (NGS).

Our custom-built proprietary bioinformatics pipeline was used for data analysis. We use this to track mutations and sequences as they move through evolutionary steps, allowing us to detect sequences enriched in capsids that have successfully been conserved within the screen.

To select the candidates that will be moved to the next stage, we calculate the enrichment score of variants, described as:

Enrichment score = (RC Variant 1 in evolved library/TotaZ RC in evolved library)/ (RC Variant 1 in AAV packaged library /T otal RC in AAV packaged library)

*RC= NGS read counts

Top candidates with higher enrichment scores are selected within individual libraries. Where in vivo data was available (for example when the library had been injected intramuscularly in mouse) enrichment data was crosschecked with the in vivo data so that only those candidates that appeared at least once in vivo are taken forward.

Methods for in vivo validation of the capsid

Each selected capsid candidate was packaged with dual reporters that allow fluorescence detection by mScarlet or GFP reporter and luciferase activity quantification.

Those capsids that had a good performance in vitro were moved to in vivo validation. We injected the selected candidates in adult black mice (8-10 weeks). The AAVs were injected bilaterally with a volume of 10 pl at each side under general anaesthesia. For each injection, we performed three x 3.3ul deposits per injection in 4mm depth at 1.3mm intervals. Because of the angle and the depth required, the whole triceps surae (calf) will be targeted, which includes the gastrocnemius lateral and medial, and the soleus muscles. For motor neuron transduction efficiency, four weeks after AAV injection mice were perfused, and spinal cord was collected. We performed immunofluorescence for general motor neuron marker (Choline acetyltransferase, ChAT) and mScarlet or GFP (our AAV reporter). We carried out quantification of the positive cells for each marker in the L3-L6 lumbar spinal cord segments.

In cases where the resolution of immunofluorescence was insufficient, the RNAscope technique was employed to achieve a more sensitive and spatially resolved detection of targets at the transcriptional level. Animals were injected and perfused as described above. Following tissue collection, horizontal sections were prepared and processed for RNAscope in situ hybridization using probes targeting ChAT and GFP. This method enabled precise localization of target mRNA expression within the tissue sections.

For biodistribution and mRNA expression in off target organs, four weeks after AAV injection mice were perfused and liver, injected muscle and non-injected muscle were collected. Genomic DNA and total RNA were extracted from each tissue using TRIzol™ reagent according to the manufacturer's protocol. RNA samples were then treated with DNase I to eliminate genomic contamination before being reverse transcribed into cDNA using a reverse transcriptase reaction.

To assess biodistribution, GFP vector copy numbers (VCN) were quantified from extracted DNA using quantitative PCR (qPCR). GFP mRNA expression levels were evaluated from cDNA using qPCR as well. All qPCR reactions were performed using gene-specific primers, and GFP copy numbers were normalized to the housekeeping gene GAPDH to account for sample variability.

Methods for in vivo assessment of the capsids in a different route of administration IV.

Adult black mice (8-10 weeks) were injected intravenously via the tail vein with 100 pL of AAV vector candidates CM02 (SEQ ID.53) and CM43 (SEQ ID.54) with the objective of addressing safety and their biodistribution pattern if they were to be injected intravenously. Five weeks after AAV injection, animals were perfused, and organs were collected. We performed motor neuron transduction efficiency and GFP biodistribution as describe above

Sequences

SEQ ID.l AVSPLHKNENVA

SEQ ID.2 AASSQSKPRATQPPVA

SE ID.3 HYFKDQY

SE ID.4 EVGNMNQ

SE ID.5 TIGCYDG

SE ID.6 FPDGRYW

SE ID.7 RGSRMTTNIYLNSS

SEQ ID.8 HQFNNIAKQVASNWYNRQIERSSRTQG SEQ ID.9 AHQFNNIAKLMA

SEQ ID.10 WVSARMA

SEQ ID.ll VASNWYNRQE

SEQ ID.12 NICKLVCSNW

SEQ ID.13 VASNWTNRQI

SEQ ID.14 VMTTNIYLNS

SEQ ID.15 VMSVLLVATA

SEQ ID.16 WYNRQIERSS

SEQ ID.17 ILSTLWKYRC

SEQ ID.18 VMTTVIYLVS

SEQ ID.19 ACQSQSQWRC

SEQ ID.20 GSVMTTNIWL

SEQ ID.21 RGSVMTTNIY

SEQ ID.22 LNKLSTLWKY

SEQ ID.23 KDVHHNI

SEQ ID.24 SGTQNFE

SEQ ID.25 GCTGTGAGCCCTCTGCACAAGAATGAGAACGTCGCC

SEQ ID.26 GCCGCTAGCTCACAATCTAAGCCAAGGGCAACACAGCCCCCTGTGGCCA

SEQ ID.27 CATTATTTTAAGGATCAGTAT

SEQ ID.28 GAGGTGGGGAATATGAATCAG

SEQ ID.29 ACGATTGGGTGTTATGATGGT

SEQ ID.30 TTTCCGGATGGGCGGTATTGG

SEQ ID.31 AGAGGCAGCAGGATGACCACAAATATCTACCTGAACTCTTCC

SEQ ID.32

GCCACCAATTCAATAACATCGCTAAGCAGGTGGCCAGCAATTGGTACAACAGGCAGATTGAGCGGTCCTCTAGA

ACCCAGG SEQ ID.33 GCACACCAGTTCAATAACATCGCTAAGCTGATGGCC

SEQ ID.34 TGGGTGAGTGCTAGGATGGCT

SEQ ID.35 GTGGCAAGCAATTGGTACAACAGGCAGGAA

SEQ ID.36 AACATCTGTAAGCTGGTGTGCAGCAATTGG

SEQ ID.37 GTGGCTAGCAATTGGACCAACAGACAGATC

SEQ ID.38 GTGATGACAACCAACATCTACCTGAATAGC

SEQ ID.39 GTTATGAGCGTACTGCTCGTGGCAACCGCC

SEQ ID.40 TGGTACAACAGGCAGATCGAGAGAAGCTCC

SEQ ID.41 ATCCTGAGCACCCTCTGGAAGTACAGGTGC

SEQ ID.42 GTGATGACCACAGTTATCTACCTGGTCAGC

SEQ ID.43 GCCTGTCAGAGCCAGTCCCAATGGAGATGC

SEQ ID.44 GGCAGCGTGATGACCACAAACATCTGGCTG

SEQ ID.45 AGGGGCAGCGTGATGACAACCAACATCTAC

SEQ ID.46 AGGGGCAGCGTGATGACCACAAACATCTAC

SEQ ID.47 CTCAACAAACTGAGCACCCTTTGGAAGTAC

SEQ ID.48 AAGGATGTTCATCATAATATT

SEQ ID.49 TCTGGGACTCAGAATTTTGAG

SEQ ID.50

>AAV1 CAP WT

MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALE

HDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSP

QEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNA

SGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQR

LINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYG

YLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSA

QNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDE

DKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSTDPATGDVHAMGALPGMV

WQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIE

WELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL SEQ ID.51

>AAV2 CAP WT

MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEH DKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEP DSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSG NWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLIN NNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQ.VFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYL TLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQ SRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEK FFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQD RDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQ KENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

SEQ ID.52

>AAV6 CAP WT

MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALE HDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSP QEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNA SGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQR LINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYG YLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGS AQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDD KDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNLQSSSTDPATGDVHVMGALPGMV WQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIE WELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL

SEQ ID.53

>MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLD KGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQ AKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDAD SVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVI TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQ.VFTDSEYQLPYVLGSAHQG CLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPF HSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPG PCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVL IFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNAVSPLHKNENVAR QAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILI KNTPVPANPSTTFSAAKFASFITQYSTGQ.VSVEIEWELQKENSKRWNPEIQYTSNYNKSV NVDFTVDTNGVYSEPRPIGTRYLTRNL

SEQ ID.54 >MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSE SVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVI TTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRL INNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQ GCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVP FHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGIVISVQPKNWLP GPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPIVISGV MIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSAVSPLHKNENVA TDPATGDVHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLIVIGGFGLKNPPPQIL IKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKS ANVDFTVDNNGLYTEPRPIGTRYLTRPL

SEQ ID.55

>MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLD KG E PVN E AD AAALE H DKAYD RQLDSG D N PYLKYN HAD AE FQE RLKE DTS FGG N LG R AVFQ AKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDAD SVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWIVIGDRVI TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQG CLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPF HSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPG PCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVL IFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNAASSQSKPRATQP PVARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPP QILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNY NKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

SEQ ID.56

>MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLD KG E PVN E AD AAALE H DKAYD RQLDSG D N PYLKYN HAD AE FQE RLKE DTS FGG N LG R AVFQ AKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDAD SVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWIVIGDRVI TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQG CLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPF HSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPG PCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVL IFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRGSRMTTNIYLNS SRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQI LIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNK SVNVDFTVDTNGVYSEPRPIGTRYLTRNL

SEQ ID.57

>MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLD KGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQ AKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDAD SVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWIVIGDRVI TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQG CLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPF HSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPG PCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVL IFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNAHQFNNIAKLMAR QAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILI KNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSV NVDFTVDTNGVYSEPRPIGTRYLTRNL

SEQ ID.58

>MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLD KG E PVN E AD AAALE H DKAYD RQLDSG D N PYLKYN HAD AE FQE RLKE DTS FGG N LG R AVFQ AKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDAD SVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWIVIGDRVI TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQG CLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPF HSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPG PCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVL IFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNWVSARMARQAATA DVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPV PANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFT VDTNGVYSEPRPIGTRYLTRNL

SEQ ID.59

>MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLD KG E PVN E AD AAALE H DKAYD RQLDSG D N PYLKYN HAD AE FQE RLKE DTS FGG N LG R AVFQ AKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDAD SVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWIVIGDRVI TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQG CLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPF HSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPG PCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVL IFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNVASNWYNRQERQA ATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKN TPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNV DFTVDTNGVYSEPRPIGTRYLTRNL

SEQ ID.60 >MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLD KG E PVN E AD AAALE H DKAYD RQLDSG D N PYLKYN HAD AE FQE RLKE DTS FGG N LG R AVFQ AKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDAD SVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWIVIGDRVI TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQG CLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPF HSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPG PCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVL IFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNNICKLVCSNWRQA ATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKN TPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNV DFTVDTNGVYSEPRPIGTRYLTRNL

SEQ ID.61

>MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLD KG E PVN E AD AAALE H DKAYD RQLDSG D N PYLKYN HAD AE FQE RLKE DTS FGG N LG R AVFQ AKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDAD SVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWIVIGDRVI TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQG CLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPF HSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPG PCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVL IFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNVASNWTNRQIRQA ATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKN TPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNV DFTVDTNGVYSEPRPIGTRYLTRNL

SEQ ID.62

>MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLD KG E PVN E AD AAALE H DKAYD RQLDSG D N PYLKYN HAD AE FQE RLKE DTS FGG N LG R AVFQ AKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDAD SVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWIVIGDRVI TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQG CLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPF HSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPG PCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVL IFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNVMTTNIYLNSRQA ATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKN TPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNV DFTVDTNGVYSEPRPIGTRYLTRNL SE ID.63

>MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLD KG E PVN E AD AAALE H DKAYD RQLDSG D N PYLKYN H AD AE FQE RLKE DTS FGG N LG R AVFQ AKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDAD SVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVI TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQG CLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPF HSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPG PCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVL IFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNVMSVLLVATARQA ATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKN TPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNV DFTVDTNGVYSEPRPIGTRYLTRNL

SE ID. 64

>MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLD KGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQ AKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDAD SVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWIVIGDRVI TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQG CLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPF HSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPG PCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVL IFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNWYNRQIERSSRQA ATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKN TPVPANPSTTFSAAKFASFITQYSTGQ.VSVEIEWELQKENSKRWNPEIQYTSNYNKSVNV DFTVDTNGVYSEPRPIGTRYLTRNL

SEQ ID.65

>MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLD KGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQ AKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDAD SVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWIVIGDRVI TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQG CLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPF HSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPG PCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVL IFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNHQFNNIAKQVASN WYNRQIERSSRTQGRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPL MGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

SEQ ID.66

>MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLD KG E PVN E AD AAALE H DKAYD RQLDSG D N PYLKYN HAD AE FQE RLKE DTS FGG N LG R AVFQ AKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDAD SVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWIVIGDRVI TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQG CLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPF HSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPG PCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVL IFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNILSTLWKYRCRQA ATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKN TPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNV DFTVDTNGVYSEPRPIGTRYLTRNL

SEQ ID.67

>MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLD KG E PVN E AD AAALE H DKAYD RQLDSG D N PYLKYN HAD AE FQE RLKE DTS FGG N LG R AVFQ AKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDAD SVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWIVIGDRVI TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQG CLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPF HSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPG PCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVL IFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNVMTTVIYLVSRQA ATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKN TPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNV DFTVDTNGVYSEPRPIGTRYLTRNL

SEQ ID.68

>MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLD KG E PVN E AD AAALE H DKAYD RQLDSG D N PYLKYN HAD AE FQE RLKE DTS FGG N LG R AVFQ AKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDAD SVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWIVIGDRVI TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQG CLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPF HSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPG PCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVL IFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNACQSQSQWRCRQA ATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKN

TPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNV

DFTVDTNGVYSEPRPIGTRYLTRNL

SEQ ID.69

>MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLD KG E PVN E AD AAALE H DKAYD RQLDSG D N PYLKYN HAD AE FQE RLKE DTS FGG N LG R AVFQ AKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDAD SVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWIVIGDRVI TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQG CLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPF HSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPG PCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVL IFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNGSVMTTNIWLRQA ATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKN TPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNV DFTVDTNGVYSEPRPIGTRYLTRNL

SEQ ID.70

>MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLD KG E PVN E AD AAALE H DKAYD RQLDSG D N PYLKYN HAD AE FQE RLKE DTS FGG N LG R AVFQ AKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDAD SVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWIVIGDRVI TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQG CLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPF HSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPG PCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVL IFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRGSVMTTNIYRQA ATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKN TPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNV DFTVDTNGVYSEPRPIGTRYLTRNL

SEQ ID.71

>MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSE SVPDPQPLGEPPATPAAVGPTTMASGGGAPIVIADNNEGADGVGNASGNWHCDSTWLGDRVI TTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRL INNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQ GCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVP FHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGIVISVQPKNWLP GPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPIVISGV MIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQHYFKDQYSSSTDPAT GDVHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLIVIGGFGLKNPPPQILIKNTP VPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDF TVDNNGLYTEPRPIGTRYLTRPL

SEQ ID.72

>MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSE SVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVI TTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRL INNNWGFRPKRLNFKLFNIQ.VKEVTTNDGVTTIANNLTSTVQ.VFSDSEYQLPYVLGSAHQ GCLPPFPADVFM IPQYGYLTLNNGSQAVGRSSFYCLEYFPSQM LRTGNNFTFSYTFEEVP FHSSYAHSQSLDRLM NPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLP GPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGV M IFGKESAGASNTALDNVM ITDEEEIKATNPVATERFGTVAVNFQSSSEVGNMNQTDPAT GDVHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTP VPANPPAEFSATKFASFITQYSTGQ.VSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDF TVDNNGLYTEPRPIGTRYLTRPL

SEQ ID.73

>MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLD KG E PVN E AD AAALE H DKAYD RQLDSG D N PYLKYN H AD AE FQE RLKE DTS FGG N LG R AVFQ. AKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDAD SVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVI TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRPKRLNFKLFNIQ.VKEVTQNDGTTTIANNLTSTVQ.VFTDSEYQLPYVLGSAHQG CLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPF HSSYAHSQSLDRLM NPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPG PCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVL IFGKQGSEKTNVDIEKVM ITDEEEIRTTNPVATEQYGSVSTNLQRGNRGSVMTTNIYRQA ATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKN TPVPANPSTTFSAAKFASFITQYSTGQ.VSVEIEWELQKENSKRWNPEIQYTSNYNKSVNV DFTVDTNGVYSEPRPIGTRYLTRNL

SEQ ID.74

>MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSE SVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVI TTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRL INNNWGFRPKRLNFKLFNIQ.VKEVTTNDGVTTIANNLTSTVQ.VFSDSEYQLPYVLGSAHQ GCLPPFPADVFM IPQYGYLTLNNGSQAVGRSSFYCLEYFPSQM LRTGNNFTFSYTFEEVP FHSSYAHSQSLDRLM NPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLP GPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGV M IFGKESAGASNTALDNVM ITDEEEIKATNPVATERFGTVAVNFQSSSTIGCYDGTDPAT GDVHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTP VPANPPAEFSATKFASFITQYSTGQ.VSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDF TVDNNGLYTEPRPIGTRYLTRPL

SEQ ID.75 >MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSE SVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVI TTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRL INNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQ.VFSDSEYQLPYVLGSAHQ GCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVP FHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLP GPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGV MIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSFPDGRYWTDPAT GDVHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTP VPANPPAEFSATKFASFITQYSTGQ.VSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDF TVDNNGLYTEPRPIGTRYLTRPL

SEQ ID.76

>MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSE SVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVI TTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRL INNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQ.VFSDSEYQLPYVLGSAHQ GCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVP FHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLP GPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGV MIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSTDPATGDVHAMG ALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPA EFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGL YTEPRPIGTRYLTRPL

SEQ ID.77

>MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSE SVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVI TTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRL INNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQ.VFSDSEYQLPYVLGSAHQ GCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVP FHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLP GPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGV MIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSTDPATGDVHAMG ALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPA EFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGL YTEPRPIGTRYLTRPL

SEQ ID.78

>MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSE SVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVI TTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRL INNNWGFRPKRLNFKLFNIQ.VKE TTNDG TTIANNLTSTVQ.VFSDSEYQLPYVLGSAHQ GCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVP FHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLP GPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGV MIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNLQKDVHHNISSSTDPAT GDVHVMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTP VPANPPAEFSATKFASFITQYSTGQ.VSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDF TVDNNGLYTEPRPIGTRYLTRPL

SEQ ID.79

>MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSE SVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVI TTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRL INNNWGFRPKRLNFKLFNIQ.VKEVTTNDGVTTIANNLTSTVQ.VFSDSEYQLPYVLGSAHQ GCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVP FHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLP GPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGV MIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNLQSSGTQNFESSTDPAT GDVHVMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTP VPANPPAEFSATKFASFITQYSTGQ.VSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDF TVDNNGLYTEPRPIGTRYLTRPL

SEQ ID.80

>MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSE SVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVI TTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRL INNNWGFRPKRLNFKLFNIQ.VKE TTNDG TTIANNLTSTVQ.VFSDSEYQLPYVLGSAHQ GCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVP FHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLP GPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGV MIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNLQAVSPLHKNENVASSS TDPATGDVHVMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIL IKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKS ANVDFTVDNNGLYTEPRPIGTRYLTRPL

SEQ ID.81

>MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSE SVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVI TTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRL INNNWGFRPKRLNFKLFNIQ.VKE TTNDG TTIANNLTSTVQ.VFSDSEYQLPYVLGSAHQ GCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVP FHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLP GPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGV MIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNLQRGSRMTTNIYLNSSS SSTDPATGDVHVMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQ ILIKNTPVPANPPAEFSATKFASFITQYSTGQ.VSVEIEWELQKENSKRWNPEVQYTSNYA KSANVDFTVDNNGLYTEPRPIGTRYLTRPL

SEQ ID.82

>MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSE SVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVI TTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRL INNNWGFRPKRLNFKLFNIQ.VKEVTTNDGVTTIANNLTSTVQ.VFSDSEYQLPYVLGSAHQ GCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVP FHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLP GPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGV MIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNLQAHQFNNIAKLMASSS TDPATGDVHVMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIL IKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKS ANVDFTVDNNGLYTEPRPIGTRYLTRPL

SEQ ID.83

>MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSE SVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVI TTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRL INNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQ.VFSDSEYQLPYVLGSAHQ GCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVP FHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLP GPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGV MIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSRGSRMTTNIYLN SSTDPATGDVHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQ ILIKNTPVPANPPAEFSATKFASFITQYSTGQ.VSVEIEWELQKENSKRWNPEVQYTSNYA KSANVDFTVDNNGLYTEPRPIGTRYLTRPL

SEQ ID.84

>MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSE SVPDPQPLGEPPATPAAVGPTTMASGGGAPIVIADNNEGADGVGNASGNWHCDSTWLGDRVI TTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRL INNNWGFRPKRLNFKLFNIQ.VKEVTTNDGVTTIANNLTSTVQ.VFSDSEYQLPYVLGSAHQ GCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVP FHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGIVISVQPKNWLP GPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPIVISGV MIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSAASSQSKPRATQ PPVATDPATGDVHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLIV1GGFGLKNPP PQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSN YAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL

SEQ ID.85

>MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSE SVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVI TTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRL INNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQ.VFSDSEYQLPYVLGSAHQ GCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVP FHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLP GPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGV MIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNLQAASSQSKPRATQPPV ASSSTDPATGDVHVMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPP PQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSN YAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL

SEQ ID.86

>MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSE SVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVI TTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRL INNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQ.VFSDSEYQLPYVLGSAHQ GCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVP FHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLP GPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGV MIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSHQFNNIAKQVAS NWYNRQIERSSRTQGTDPATGDVHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSP LMGGFGLKNPPPQIUKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSK RWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL

SEQ ID.87

>MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSE SVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVI TTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRL INNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQ.VFSDSEYQLPYVLGSAHQ GCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVP FHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLP GPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGV MIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNLQHQFNNIAKQVASNWY NRQIERSSRTQGSSSTDPATGDVHVMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSP LMGGFGLKHPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSK RWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL

SEQ ID.88 >MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSE SVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVI TTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRL INNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQ.VFSDSEYQLPYVLGSAHQ GCLPPFPADVFM IPQYGYLTLNNGSQAVGRSSFYCLEYFPSQM LRTGNNFTFSYTFEDVP FHSSYAHSQSLDRLM NPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLP GPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGV M IFGKESAGASNTALDNVM ITDEEEIKATNPVATERFGTVAVNLQHYFKDQYSSSTDPAT GDVHVMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTP VPANPPAEFSATKFASFITQYSTGQ.VSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDF TVDNNGLYTEPRPIGTRYLTRPL

SEQ ID.89

>MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSE SVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVI TTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRL INNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQ.VFSDSEYQLPYVLGSAHQ GCLPPFPADVFM IPQYGYLTLNNGSQAVGRSSFYCLEYFPSQM LRTGNNFTFSYTFEDVP FHSSYAHSQSLDRLM NPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLP GPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGV M IFGKESAGASNTALDNVM ITDEEEINATNPVATERFGTVSVNLHSSSTDPATGDVLVMG ALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPA EFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGL YTEPRPIGTRYLTRPL

SEQ ID.90

>MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLD KG E PVN E AD AAALE H DKAYD RQLDSG D N PYLKYN H AD AE FQE RLKE DTS FGG N LG R AVFQ. AKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDAD SVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVI TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQ.VFTDSEYQLPYVLGSAHQG CLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPF HSSYAHSQSLDRLM NPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPG PCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVL IFGKQGSEKTNVDIEKVM ITDEEEIRTTNPVATEQYGSVSTNLQRGNLNKLSTLWKYRQA ATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKN TPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNV DFTVDTNGVYSEPRPIGTRYLTRNL

Claims

Claims

1. An amino acid sequence insert according to any one of SEQ ID.l to SEQ ID.24 for insertion into an AAV capsid peptide to create a targeted AAV capsid peptide; wherein, when the targeted AAV capsid peptides assemble into a targeted AAV capsid, the targeted AAV capsid has increased tropism for motor neurons relative to an AAV capsid without the insert.

2. Use of an amino acid sequence insert according to any one of SEQ ID.l to SEQ ID.24 as an insertion into an AAV capsid peptide to generate a targeted AAV capsid peptide which assembles into a targeted AAV capsid with increased tropism for motor neurons relative to an AAV capsid without the insert.

3. A targeted AAV capsid peptide comprising the amino acid sequence insert according to any one of SEQ ID.l to SEQ ID.24 inserted into an AAV capsid peptide.

4. The targeted AAV capsid peptide according to claim 3 wherein AAV capsid peptide is an AAV1, AAV2, or AAV6 serotype capsid peptide.

5. The targeted AAV capsid peptide according to claim 4, the AAV capsid peptide having an amino acid sequence according to any one of SEQ ID.50 to SEQ ID.52.

6. The targeted AAV capsid peptide according to any one of claims 3 to 5 wherein the amino acid sequence insert is inserted at position, Q585/S586 or S588/T589 in SEQ ID.50, N587/R588 in SEQ ID.51, or Q585/S586, S586/S587, or S587/S588 in SEQ ID.52 or the equivalent position thereof.

7. The targeted AAV capsid peptide according to claim 6 having an amino acid sequence selected from SEQ ID.53 to SEQ ID.90.

8. A targeted AAV capsid comprising one or more targeted AAV capsid peptides according to claim 3 to 7.

9. A targeted AAV viral vector comprising the targeted AAV capsid according to claim 8 and a nucleotide-encoded payload.

10. The targeted AAV viral vector according to claim 9 wherein the nucleotide-encoded payload encodes a therapeutic peptide.

11. The targeted AAV viral vector according to claim 9 or claim 10 for use in the treatment of a disease caused by a genetic mutation.

12. The targeted AAV viral vector according to any one of claims 9 to 11 for use in the treatment of a disorder associated with motor neurons.

13. The targeted AAV viral vector for use according to claim 12 wherein the disorder is spasticity.

14. A nucleotide sequence encoding the amino acid sequence according to any one of SEQ ID.l to SEQ ID.24 or SEQ ID.53 to SEQ ID.90 for use in generating a targeted AAV capsid with increased tropism for motor neurons.

15. The nucleotide sequence according to claim 14 comprising the sequence of SEQ ID.25 to SEQ ID.49.

16. The targeted AAV capsid peptide according to any one of claims 3 to 11 wherein, when the serotype is AAV6, the peptide sequence may further comprise a point mutation at L584F and/or V598A of SEQ ID.52, or equivalent residues thereof.